Optical Image Scanning Microscope

The invention describes a simple way of performing Image Scanning Microscopy (ISM) purely optically. It is not necessary to reconstruct the final image from a multitude of individual acquisitions. Instead, the final image is projected directly onto the camera chip. The method is suitable for confocal microscopy as well as for 2-photon microscopy.

Challenge

Image Scanning Microscopy was developed during the last years and improves the lateral resolution by a factor of 1.6 in comparison with confocal Laser Scanning Microscopy (cLSM). The principal of the method is similar to how the improved resolution in structured illumination microscopy is obtained.

Previously, the disadvantage of ISM was, that for each scan position of the excitation laser an individual image on the camera chip had to be acquired. Subsequently, the final image was calculated by combining all sub-images. Although with this approach ISM was already capable of reaching a significantly higher resolution than cLSM setups, it was also much slower.

New developments have shown that optical ISM without computer aided reconstruction is possible (Optical Photon Reassignment – OPRA). Unfortunately, OPRA demands quite complex optics and it is difficult to upgrade existing cLSM setups. In addition, excitation and emission light pass through the same optics which makes it difficult to apply the method in 2-photon microscopy.

Our Solution

The newly developed, optical ISM method overcomes the above mentioned disadvantages by producing the final image directly on the camera chip. Neither the computer aided reconstruction nor the readout of the camera chip for each scan position are necessary. Consequently, the new method is significantly faster while providing the same benefits in terms of resolution.

The new method is a purely optical realization of the ISM idea. However, in contrast to previous realizations, ISM is here performed through increasing the distance between two scan positions on the camera chip by a factor of 2. To obtain the desired result, a second scan mirror can be used. But it is even possible to avoid using this additional scan mirror. The first scan mirror, which is anyway already available, can be used instead, if the emission light is passed through a 2f-arrangement. The method can thus be applied with a small amount of additional optical components. It is ideally suited to upgrade existing cLSM systems.

Furthermore, it is possible to have separated light pathways for excitation and emission light. Therefore, the optical components in the emission pathway can be adjusted completely to the emission wavelength. The newly developed ISM realization is thus also suitable for 2-photon microscopy.

Advantages

The resolution improvement by a factor of 1.6 typically gained with ISM is obtained without limiting the versatility of the setup for example in terms of usable fluorophores. In addition, the new ISM realization offers the following advantages:

Optical realization, the final image is created directly on the camera chip. => High frame rates.

Only very few additional optical components are necessary. => Existing cLSM setups can be easily upgraded without limiting their normal capabilities.

No second scan mirror necessary. => Avoids synchronization problems between the scan mirrors.